Alliance 2695 system

Manufactured by Waters Corporation

Sourced in United States, France

The Alliance 2695 system is a high-performance liquid chromatography (HPLC) instrument manufactured by Waters Corporation. It is designed for analytical and preparative chromatographic separations, providing precise control and monitoring of various parameters such as flow rate, gradient composition, and sample injection. The Alliance 2695 system is a versatile platform suitable for a wide range of applications in the field of analytical chemistry and biochemistry.

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25 protocols using alliance 2695 system

Quantifying Decitabine in Biological Samples by LC-MS/MS

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A liquid chromatography method using tandem mass spectrometry (LC-MS/MS) was developed to quantify decitabine (C12)₂. It was performed on an Alliance^® 2695 system (Waters, Saint-Quentin-en-Yvelines, France) with an Uptisphere C18-ODB 150×2.0 mm, 5 µm column (Inter-chrom, Montluçon, France). The mobile phase consisted of a phase A (0.1% formic acid in acetonitrile) and a phase B (0.1% formic acid in methanol) in an isocratic mode (A:B 70:30 v/v). The flow rate was set to 0.4 mL/min for a run time of 6 minutes. The injection volume was 10 µL. The total HPLC effluent was directed into a Quattro Micro^® triple quadrupole mass spectrometer (Waters). Ionization was achieved with an electrospray in the positive ion mode. The mass spectrometer was operated in the multiple reaction monitoring mode. The (M–H)+m/z transition for decitabine (C12)₂ was 593.3>113.1. The entire system was controlled by Masslynx^® software (Waters).
Calibration curves were obtained after solubilizing decitabine (C12)₂ in methanol to obtain a stock solution of 0.5 mg/mL. A sufficient quantity of the stock solution was then diluted in methanol to obtain a calibration curve between 2.5 and 500 ng/mL (seven plots were used). A calibration curve was performed on each day of the analysis.

Briot T., Roger E., Bou Haidar N., Bejaud J., Lautram N., Guillet C., Thépot S., Legeay S, & Lagarce F. (2019). Di-O-lauroyl-decitabine-lipid nanocapsules: toward extending decitabine activity. International Journal of Nanomedicine, 14, 2091-2102.

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Quantification of Anthocyanins and Flavonols

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Extract anthocyanin and flavonol quantification was performed in a Waters Alliance 2695 system equipped with a W2690/5 separation module unit, a DAD detector, and a 4.6 × 150 mm, 5 μm C18 Waters Spherisorb column at 40 °C. The chromatographic separation was carried out using the same gradient system that LC-MS. Quantification was achieved by external standardization, using pure commercial standards of glycosylated anthocyanins and glycosylated flavonols purchased from Extrasynthese (Genay, France). Stock solutions of all standard anthocyanin compounds were prepared in methanol-HCl 0.1% while flavonols in ethanol. Flavonoids calibration protocols were prepared by diluting stock solutions in ranges of 0.01 to 0.2 mM; calibration curves plotted peak areas versus concentrations (R² = 0.99). Results are expressed as milligrams of compound per gram extract, as shown in supplemental Table 2.

Calfío C, & Huidobro-Toro J.P. (2019). Potent Vasodilator and Cellular Antioxidant Activity of Endemic Patagonian Calafate Berries (Berberis microphylla) with Nutraceutical Potential. Molecules, 24(15), 2700.

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HPLC-DAD Analysis of Compounds

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HPLC-DAD analysis was performed on a Waters Alliance 2695 system (Waters, Milford, MA, USA) consisting of a 2695 module and a 2998 DAD detector. A YMC HPLC C30 column (150 × 4.6 mm, 3 μm, YMC Co., LTD., Kyoto, Japan) was applied for the separation. The mobile phase was composed of A (methanol/MTBE/water, 81:15:4, v/v/v) and B (methanol/MTBE/water, 16:80:4, v/v/v) with the following gradient elution: 0–7 min, 80%–50% A; 7–19 min, 50%–22% A; 19–22 min, 22% A; 22–37 min, 22%–0% A; 37–40 min, 0%–80% A. The column temperature was set at 25 °C. The flow rate was set at 0.4 mL/min. The injection volume was 10 μL, and the detection wavelength was 450 nm.

Lu Y., Guo S., Zhang F., Yan H., Qian D.W., Wang H.Q., Jin L, & Duan J.A. (2019). Comparison of Functional Components and Antioxidant Activity of Lycium barbarum L. Fruits from Different Regions in China. Molecules, 24(12), 2228.

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COLI Concentration Quantification via LC-MS/MS

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The determination of COLI plasma concentrations was performed based on a validated LC-MS/MS method previously described [17 (link),18 (link),24 (link)]. The chromatography was performed on an Alliance 2695 system (Waters, France) with a Jupiter 300-Å column (5.0 µm, 50 mm, 2.0-mm i.d.; Waters, St.-Quentin en Yvelines, France) and a mobile phase (flow rate 0.2 mL/min) consisting of 0.1% (v/v) formic acid in acetonitrile–0.1% formic acid in water (25:75 v/v). The mass spectrometer Micromass Quattro microAPI (Waters, France) was used in the positive/ion mode. The calibration standard curve was prepared with seven samples in rat plasma with COLI base concentrations ranging between 0.0097 and 10 µg·mL⁻¹. For analysis of COLI in BALF and in the medium used for the COLI transport experiments, 150 µL of sample was mixed with 100 µL of plasma and analyzed as for plasma.

Tewes F., Brillault J., Gregoire N., Olivier J.C., Lamarche I., Adier C., Healy A.M, & Marchand S. (2020). Comparison between Colistin Sulfate Dry Powder and Solution for Pulmonary Delivery. Pharmaceutics, 12(6), 557.

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Purification and Characterization of Organic Compounds

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All reagents were purchased from Acros, Aldrich, Alfa Aesar or Merck and used without further purification. Thin layer chromatography was performed on silica gel 60 F254 aluminium sheets 20 × 20 cm (Merck), as eluent using (20% EtOAc/hexane). The purities of compounds were determined by high performance liquid chromatography on a Waters Alliance 2695 system and Waters 2489 ultraviolet–vis detector equipped with Alltima C18 column (5 µm, 4.6 × 150 mm, Grace) using a gradient elution with acetonitrile/phosphoric acid (0.1%) in water, at a flow rate of 1 ml min⁻¹. Peak areas were determined electronically with a Waters Empower 2 chromatography data system.

Petrova M., Muhamadejev R., Vigante B., Duburs G, & Liepinsh E. (2018). Intramolecular hydrogen bonds in 1,4-dihydropyridine derivatives. Royal Society Open Science, 5(6), 180088.

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Extraction and Analysis of Green Tea Catechins

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Fresh green tea (Camellia sinensis) leaves were collected in the spring from the Osulloc Tea Garden in Jeju, Korea and dried. Dried C. sinensis leaves were subjected to extraction twice with 50% aqueous ethanol and incubation at 100°C (1.2 atm) under aqueous conditions for 5 h to obtain GCG-GTE. The catechin contents in GCG-GTE were measured and analyzed using high-performance liquid chromatography (HPLC) with a photodiode array (PDA) detector (Alliance 2695 system, Waters) and a Thermo Syncronis C18 column (250 × 4.6 mm, I.D., 5 μm; Thermo Fisher Scientific Inc.) GCG-GTE contained almost the same amount of epicatechins and epicatechin epimers (Table 1).

Cha J., Kim H.S., Kwon G., Cho S.Y, & Kim J.M. (2023). Acute effects of (–)-gallocatechin gallate-rich green tea extract on the cerebral hemodynamic response of the prefrontal cortex in healthy humans. Frontiers in Neuroergonomics, 4, 1136362.

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Size Exclusion Chromatography of Protein Complexes

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Size exclusion chromatography was performed with a Superdex 200 increase 10/300 GL column (Cytiva) using an Alliance 2695 system (Waters). Light scattering (LS) and refractive index (RI) were measured using a DAWN HELEOS II detector (Wyatt Technology) and 2414 RI detector (Waters), respectively. Before SEC-MALS analysis, the column was equilibrated at 293 K with 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl. Bn86287 (3.7 mg/ml, 30 μl) and all2909 (4.9 mg/ml, 30 μl) were injected under the buffer flow rate of 0.5 ml/min. Data were processed with the ASTRA 6.1 software (Wyatt Technology).

Hirakawa Y., Senda M., Fukuda K., Yu H.Y., Ishida M., Taira M., Kinbara K, & Senda T. (2021). Characterization of a novel type of carbonic anhydrase that acts without metal cofactors. BMC Biology, 19, 105.

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Purification and Characterization of Peptide by HPLC-MS/MS

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To purify the peptide, semi preparative high-performance liquid chromatography (HPLC) from Waters instrument (Waters, Saint-Quentin-en-Yvelines, France) was carried out, according to the protocol previously published by Guyon et al., 2019 (link). Specifically, to elute the nude-NFL-peptide a H₂O/ACN gradient was applied (90/10, v/v; Waters) (Supplemental Table S1). The peptide was prepared at 6 mg/mL in H₂O/ACN (60/40, v/v; Waters), and the chromatographic profile was obtained (Supplemental Fig. S1).
The nude-NFL-peptide was characterized by LC-MS/MS method using an Alliance® 2695 system (Waters). The same parameters were applied as previously described by Guyon et al., 2019 (link). The column used was an Uptisphere C18 50 dB (Interchim, Montluçon, France). Depending on the peptide analyzed, a H₂O/ACN specific gradient elution was applied (95/5, v/v; Waters; Supplemental Table S2), and the peptide was dissolved at 10 mg/mL in water/ACN. The m/z applied was 200–2000 range (full scan acquisition and the cone ramp 30 was used to detect the nude-NFL-peptide; Supplemental Fig. S2).

Griveau A., Alnemeh-Al Ali H., Jourdain M.A., Dupont A, & Eyer J. (2022). Characterization and quantification of the interaction between the NFL-TBS.40‐63 peptide and lipid nanocapsules. International Journal of Pharmaceutics: X, 4, 100127.

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HPLC-PDA Analysis of HTP-GTE

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HTP-GTE was analyzed by HPLC-PDA (Alliance 2695 system, Waters, USA) using a ThermoSyncronis C18 column (250 × 4.6 mm, I.D., 5 μm; Thermo Fisher Scientific Inc.). The mobile phases were 0.1% acetic acid in water for solvent A and acetonitrile for solvent B. The gradient elution was 90% A + 10% B at 0–10 min, 85% A + 15% B at 10–30 min, 80% A + 20% B at 30–53 min, 5% A + 95% B at 53–55 min, 90% A + 10% B at 55–60 min with a flow rate of 1.0 mL/min. The sample volume for injection was 10 μl, and the measurement was carried at the UV wavelength of 280 nm.

Ahn J.W., Kim S., Ko S., Kim Y.H., Jeong J.H, & Chung S. (2022). Modified (−)-gallocatechin gallate-enriched green tea extract rescues age-related cognitive deficits by restoring hippocampal synaptic plasticity. Biochemistry and Biophysics Reports, 29, 101201.

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Characterization of Organic Compounds by NMR and Mass Spectrometry

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One-dimensional ¹H and ¹³C NMR spectra were recorded on a Varian 400 Mercury spectrometer at 400 MHz (for ¹H nuclei) and 100 MHz (for ¹³C nuclei). The chemical shifts are presented in parts per million (ppm). Residual protons of deuterated solvents were used as internal standard for ¹H NMR spectra (CDCl₃: δ 7.26 ppm, DMSO-d₆: δ 2.50). The deuterated solvent signals were used as internal standards for ¹³C NMR spectra.
Low-resolution mass spectra (MS) were recorded on a Waters ACQUITY UPLC system equipped with a BEH C₁₈ column connected to a Waters SQ Detector 2 operating in the ESI positive ion mode. Elemental analyses were performed on an Elemental Combustion System ECS 4010 (Costech Instruments) at the Laboratory of Chromatography, Latvian Institute of Organic Synthesis. All target compounds had >95% purity. The purity of each compound was determined by HPLC on a Waters Alliance 2695 system equipped with an Altima C₁₈ column, 5 µM, 4.6 × 150 mm and a Waters 2489 UV-VIS detector, using a gradient elution with acetonitrile/H₃PO₄ (0.1%) in water at a flow rate of 1 mL/min.

Bisenieks E., Vigante B., Petrovska R., Turovska B., Muhamadejev R., Soloduns V., Velena A., Pajuste K., Saso L., Klovins J., Duburs G, & Mandrika I. (2021). The Specificity and Broad Multitarget Properties of Ligands for the Free Fatty Acid Receptors FFA3/GPR41 and FFA2/GPR43 and the Related Hydroxycarboxylic Acid Receptor HCA2/GPR109A. Pharmaceuticals, 14(10), 987.

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